The challenging retention of polar organic acids in reversed phase conditions is accomplished using a unique multi-mode stationary phase.
The challenging retention of polar organic acids in reversed phase conditions is accomplished using a unique multi-mode stationary phase. Retention characteristics are compared with a standard ODS stationary phase highlighting the value of additional selectivities in development of methods for organic acids.
Analysis of organic acids in a mixture has been challenging due to the high polarity of these compounds. Typically, organic acids are analyzed with both polar and nonpolar analytes using reversed phase ODS columns. This can be difficult because their hydrophobic moieties may not differ from analyte to analyte or their polar functional groups may override this interaction. Therefore, the use of a multi-mode column like Scherzo SM-C18, which utilizes weak anion and weak cation exchange ligands in addition to hydrophobic ODS ligands provides additional ionic retention mechanisms, allowing for superior retention of organic acids.
A 4 µL sample at 3.8 to 6.2 µg concentration was injected onto a Unison UK-C18 and a Scherzo SM-C18 column (250 × 3.0 mm, 3 µ). Eluent was analyzed using ELSD detection (spray chamber 19 °C, drift tube 43 °C). Further experimental details are provided as an insert in Figure 1.
Figure 1: Comparison of retention for several organic acids on a Scherzo SM-C18 multi-mode versus traditional ODS stationary phase.
Direct comparison of two different reversed phase columns shows improved retention of organic acids on Scherzo SM-C18 versus a traditional ODS column. In this experiment the variables were reduced to only a change in stationary phase where column dimension, particle size, and gradient conditions were identical.
In this case, retention of all organic acids tested showed an increase in retention time except for succinic acid, which was reduced by 1.7 min. This could be due to a dominance of hydrophobic retention for this molecule, which would be slightly reduced in Scherzo compared to the Unison column because of a change in carbon load. The most dramatic increase was seen with maleic acid which had an additional 20.4 min of retention on Scherzo SM-C18. This could be due to the fact that maleic acid has a high dipole moment charge (7.2 debyes) because of its assymetric cis configuration. This is in contrast to its trans-isomer fumaric acid which is symmetrical and therefore no initial dipole moment.
Overall peak shape was also improved particularly for citric and maleic acid, where these analytes showed split peaks (7.6 and 7.9, 5.9 and 6.5, respectively) on a traditional ODS column.
In summary, use of a multi-mode stationary phase like Scherzo SM-C18 has several advantages over traditional ODS column in the analysis of organic acids. With better retention of these polar compounds, this column can provide better resolution in reversed phase conditions by the addition of ion exchange retention mechanisms. These additional selectivities make it possible to separate organic acids in reverse phase conditions which previously was not possible by traditional ODS stationary phases.
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